Bioorganic & Medicinal Chemistry 18 (2010) 5032–5038

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier .com/locate /bmc

Synthesis of a series of caffeic acid phenethyl amide (CAPA) fluorinatedderivatives: Comparison of cytoprotective effects to caffeic acid phenethylester (CAPE)

John Yang a, Gwendolyn A. Marriner c, Xinyu Wang a, Phillip D. Bowman b, Sean M. Kerwin c,*,Salomon Stavchansky a,*

a Division of Pharmaceutics, College of Pharmacy, The University of Texas, Austin, TX 78712, USAb US Army Institute of Surgical Research, San Antonio, TX 78234, USAc Division of Medicinal Chemistry and Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 March 2010Revised 27 May 2010Accepted 31 May 2010Available online 11 June 2010

Keywords:Oxidative stressHydrogen peroxideIschemia/reperfusion injuryCAPE

0968-0896/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.bmc.2010.05.080

* Corresponding authors. Tel.: +1 512 471 1407; fax+1 512 471 5074; fax: +1 512 232 2606 (S.M.K.).

E-mail addresses: skerwin@mail.utexas.edu (S.M.l.utexas.edu (S. Stavchansky).

A series of catechol ring-fluorinated derivatives of caffeic acid phenethyl amide (CAPA) were synthesizedand screened for cytoprotective activity against H2O2 induced oxidative stress in human umbilical veinendothelial cells (HUVEC). CAPA and three fluorinated analogs were found to be significantly cytoprotec-tive when compared to control, with no significant difference in cytoprotection between caffeic acidphenethyl ester (CAPE) and CAPA.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Oxidative stress plays a significant role in the development of avariety of disease states such as inflammation, ischemia reperfu-sion injury (I/R), and cardiovascular complications.1–4 Efforts toameliorate oxidative stress have led to the discovery of a numberof natural and novel compounds that have antioxidative propertiesor are able to induce genes with the downstream effect of counter-acting oxidative stress.5,6

Caffeic acid phenethyl ester (CAPE), a plant polyphenolic con-centrated in honeybee propolis, has been found to be biologicallyactive in a variety of pathways including cytoprotection againstoxidative stress. CAPE has been observed to exhibit anti-inflamma-tory,7 anti-viral,8 anti-carcinogenic,9 and immunomodulatory10 ef-fects as well as protection against I/R injury in vivo.11,12 CAPE hasalso been investigated for its antioxidative and radical scavengingproperties.13 Previous studies have reported the synthesis andinvestigation of catechol ring-fluorinated derivatives of CAPE withregard to cytoprotective ability against oxidative stress in vitro.14

ll rights reserved.

: +1 512 471 7474 (S.S.), tel.:

Kerwin), stavchansky@mai-

Cytoprotection to oxidative stress by CAPE and analogs has beencorrelated with their ability to up-regulate heme oxygenase 1(HMOX 1) gene expression.15,16 Despite demonstrating significantcytoprotection against oxidative damage both in vivo and in vitro,CAPE is known to be readily hydrolyzed in plasma.17,18 Studieshave suggested that esterase activity in blood and cells is respon-sible for the rapid degradation of CAPE.18 Pharmacokinetic studiesusing a rat model have also shown that CAPE is cleared rapidlyafter intravenous administration to rats.19

The purpose of this study was to synthesize and investigate thecytoprotective activity of a series of amide derivatives of CAPE andpreviously reported CAPE analogs. Amides are generally associatedwith higher hydrolytic energies of activation compared to esters.Previous work on CAPA has described its ability to act as an anti-oxidant against lipid peroxidation20 as well as a potential anti-inflammatory agent through its inhibition of 5-lipoxygenase.21

CAPA has also been shown to exhibit significant radical scavengingactivity using a 2,2-diphenyl-1-picrylhydrazyl assay.22 Althoughvarious CAPA analogues have been investigated for both radicalscavenging activity as well as a-glucosidase inhibition,23 no cate-chol ring-fluorinated CAPA analogs have been studied. The cyto-protectant ability of CAPA in vitro has also not been previouslyaddressed. Due to the importance of endothelial cells as a targetof oxidative stress in I/R injury and other vascular complications,human umbilical vein endothelial cells (HUVEC) were chosen as

Table 1Synthesis of CAPA and fluorinated CAPA analogues

H

R2R3

R4O

R5

R2R3

R4O

R5

O

O

NH

f-a4f-a3

1. TBDMSCl, imidazole, DMAP (cat) DMF, rt, 1 h

2. 2, Cs2CO3 dioxane/CHCl3, 60 °C, 18 h

3. TBAF THF, 0 °C, 5 min

Compound R2 R3 R4 R5 Yielda (%)

a (CAPA) H H H OH 14b F H H OH 7c H OH H F 15d H OMe H F 22e H H H F 8b

f F H Me OMe 63c

a Isolated overall yield from benzaldehyde 3 after column chromatography andrecrystallization.

b Isolated as �3:1 mixture of (E)-/(Z)-isomers.c Step 2 only.

J. Yang et al. / Bioorg. Med. Chem. 18 (2010) 5032–5038 5033

the model in which to study the cytoprotective effects of thesecompounds against hydrogen peroxide induced oxidative stress.

2. Results

2.1. Synthesis

CAPA and five additional fluorinated amide analogues of CAPEwere prepared using a Wittig coupling approach. The known chlo-roacetamide 1,24,25 was reacted with triphenyl phosphine to givethe phosphonium chloride 2 (Scheme 1). Wittig coupling of 2 withunprotected hydroxybenzaldehydes 3a–e proved problematic, incontrast to previous studies employing the analogous ester phos-phonium chloride.14 Thus, the hydroxybenzaldehydes 3a–e, whichwere either commercially available or obtained via demethylationof the corresponding methoxybenzaldehydes with boron tribro-mide, were first transiently protected as the t-butyldimethylsilylethers by treatment with t-butyldimethylsilyl chloride and imidaz-ole prior to Wittig coupling (Table 1). The resulting a,b-unsatu-rated amides were subjected to deprotection with TBAF, to affordCAPA and the desired amides 4b–e in modest overall yields(Table 1). For aldehydes that do not require transient protectionof the catechol functionality, the Wittig coupling proceeds in high-er yield. The dimethoxybenzaldehyde 3f was used directly in theWittig coupling to afford 4f in reasonable yield. With the exceptionof amide 4e which was isolated as a �3:1 mixture of (E)-/(Z)-iso-mers after column chromatography, the amides 4 were obtainedas >90% pure (E)-isomers after column chromatography and, for4a–c and 4f, recrystallization.

2.2. Cytotoxicity of amide derivatives compared to CAPE inHUVEC

CAPE and certain catechol ring-fluorinated CAPE analogs havebeen reported to be cytotoxic to HUVEC at higher concentrations.14

CAPA and the CAPA derivatives were screened along with CAPE fortoxicity in HUVEC. Each of the compounds were incubated withHUVEC at 10, 20, 40, and 60 lM concentrations for 24 h at 37 �C.Cell viability was measured using the CellTiter-Blue� assay andcompared to a vehicle control. Cell viability less than 90% of controlwas considered toxic. The results, shown in Figure 1 demonstratethat CAPA and amides 4b and 4d–f showed no toxicity at any ofthe tested concentrations. CAPE exhibited cytotoxicity at 40 and60 lM. The amide 4c showed cytotoxicity at all concentrations.

2.3. Cytotoxicity of H2O2 in HUVEC

Hydrogen peroxide is one of the principle reactive oxygen spe-cies produced in various vascular complications including ischemiareperfusion injury,26 and has also been used in other in vitro mod-els as an inducer of oxidative stress in endothelial cells.27,28 Todetermine a suitable dose, HUVEC were treated with H2O2 at con-centrations ranging from 0.01 to 5 mM for 1 h. Following the 1 hperiod the culture media was replaced and the cells were allowedto recover for 18 h. Cell viability was assessed with CellTiter-Blue�

following the 18 h period. The target dosage was one that reduced

HN

OPh3P

ClHN

OCl

1 2

PPh3THF, 85 °C, 72 h

82 %

Scheme 1.

cell viability to approximately 20% of control, which was providedby 2 mM H2O2. The results are shown in Figure 2.

2.4. Cytoprotection against H2O2 induced oxidative stress inHUVEC

To evaluate oxidative stress in vitro, we employed a modelusing H2O2 as the inducer of oxidative damage. HUVEC were trea-ted with CAPE or the amide derivatives 4a–f at 20 lM concentra-tion for 5 h. The cells were rinsed and then treated with 2 mMH2O2. After 1 h, the H2O2 containing medium was replaced withcell culture media and the cells were allowed to recover for 18 h.At the end of the 18 h period, cell viability was assessed with theCellTiter-Blue� Cell Viability assay and compared to cells treatedonly with vehicle and H2O2, as well as with those that were not ex-posed to H2O2. The results are shown in Figure 3. CAPA and com-pounds 4b, 4c, and 4e exhibited significant cytoprotectionagainst H2O2 when compared to vehicle only pre-treatment. CAPEwas also significantly cytoprotective against H2O2. There was nosignificant difference in cytoprotective activity between CAPEand CAPA (P >0.05).

In a dose dependent cytoprotection assay, HUVEC were treatedwith CAPE and CAPA at 1, 5, 20, 40, and 60 lM concentrations priorto the induction of oxidative stress with H2O2. The results areshown in Figure 4. The EC50 was calculated for both compoundsby linear regression using the first three data points. The EC50

was found to be 8 lM for CAPE and 2 lM for CAPA.

3. Discussion

Introducing a fluorine group on the catechol ring increases theelectronic density of the conjugated system, can decrease the inter-action with catechol methyltransferase, and may also have a signif-icant effect on receptor binding or selectivity.29 The hydroxylgroups on the CAPA catechol may contribute to the antioxidativeactivity of the compound. We were interested in seeing the effectof replacing one of these hydroxyls with a fluorine, hydrogen ormethoxy group on the cytoprotective activity of the compound.

Prior to evaluating the cytoprotective activity of CAPE and theCAPA derivatives, each compound was screened for toxicity in HU-VEC. CAPE was found to be toxic at 40 and 60 lM, in accord with

Figure 1. Toxicity of CAPE, CAPA, and CAPA derivatives toward HUVEC. Compounds were incubated in HUVEC for 24 h at 37 �C. Cell viability was determined by the AlamarBlue assay. Values are reported as a percentage of the vehicle control (0.1% DMSO).

Figure 2. Toxicity of H2O2 in HUVEC. HUVEC were incubated in culture media containing the indicated concentration of H2O2 for 1 h at 37 �C. The culture media was replacedand cells were allowed 18 h to recover, then were assessed for viability with the CellTiter-Blue�.

Figure 3. Cytoprotection of HUVEC against 2 mM H2O2 by CAPE, CAPA, and CAPAanalogues. All compound concentrations were at 20 lM. CAPE, CAPA, 4B, 4C and 4Eall showed significant cytoprotection when compared to untreated (H2O2 only)(P <0.05). CAPA derivatives 4D and 4F provided no cytoprotection.

5034 J. Yang et al. / Bioorg. Med. Chem. 18 (2010) 5032–5038

previous studies.14 There are interesting differences in cytotoxicityof the certain amide derivatives when compared to their corre-

sponding ester analogues. Amides 4e and 4f were not cytotoxicat any concentration up to 60 lM, the highest concentration exam-ined. The corresponding ester derivatives were similarly reportedto be non-cytotoxic at concentrations up to 15 lg/mL (ca.50 lM). Amide 4c was toxic at all the concentrations tested; simi-lar to the corresponding ester analogue.14 However, whereas CAPEand the esters corresponding to 4b and 4d are cytotoxic at concen-trations greater than 40 lM, CAPA and the amides 4b and 4d arenot cytotoxic even at concentrations as high as 60 lM. The originof this difference in cytotoxicity between CAPE and certain fluori-nated CAPE analogues versus CAPA and the corresponding fluori-nated CAPA analogues is not clear.

CAPE was significantly cytoprotective against H2O2 induced oxi-dative stress in HUVEC. This was also demonstrated previously in asimilar model,30 as well as a study which used menadione to gener-ate an oxidative stress.14 Four of the amide derivatives of CAPE werealso found to be significantly cytoprotective. These four compoundsall contained either one or two hydroxyl groups on the cinnamic acidphenyl ring. Although compound 4c proved to be very cytotoxic inHUVEC over a 24 h period, the toxicity is less apparent over a 5 hincubation time, as the compound was found to be significantly

Figure 4. Dose response cytoprotection relationship of CAPE and CAPA against 2 mM H2O2. Concentrations above 40 lM are cytotoxic for both CAPE and CAPA and gave lowercell viability than untreated HUVEC as shown. CAPE and CAPA showed significant cytoprotection at concentrations from 1 through 40 lM.

J. Yang et al. / Bioorg. Med. Chem. 18 (2010) 5032–5038 5035

cytoprotective against H2O2, and exhibited significantly higher cellviability over the vehicle control. While the mechanism behind thiscytoprotective activity is not completely known, it is suggested thatthe antioxidative and radical scavenging properties of the catecholgroup are correlated with the protection against H2O2. The catecholsCAPE, CAPA, 4b, and 4c all display cytoprotective effects; whereas,the monomethylated and dimethylated analogues 4d and 4f, respec-tively, were not cytoprotective. The ortho-fluorophenol 4e demon-strated intermediate cytoprotection, which may be due to theability of the ortho-fluorine substituent to stabilize the phenol radi-cal formed upon hydrogen atom donation.31 Interestingly, the cyto-protective activity for these amides is quite different from thatreported for the corresponding esters.14 The ester correspondingto 4c is not cytoprotective, despite the presence of the catechol func-tionality, and the ester corresponding to 4e is cytoprotective, despitethe lack of any free phenolic hydroxyl groups. In the dose dependentcytoprotection assay, a biphasic response was observed for bothCAPE and CAPA. The cytoprotection percentage increases from 1up to 20 lM of CAPE and CAPA, then starts to decline at 40 lM.The drop off in cytoprotection at higher CAPE concentration hadbeen attributed to CAPE’s cytotoxicity above 20 lM,13 as indicatedin Figure 1. However, a similar effect is observed for CAPA, eventhough it is not cytotoxic at 40 lM. It is unclear as to why this phe-nomenon occurs.

It is not well understood how cytoprotection is provided by pre-treatment with these cytoprotective agents. Cytoprotection proba-bly involves an interplay between direct anti-oxidant activity andindirect anti-oxidant activity through effects on the transcriptionof anti-oxidant genes.32 Studies previously performed in our grouphave shown that the cytoprotective activity of CAPE was correlatedwith the levels of the heme oxygenase 1 (HMOX 1) gene expres-sion. CAPE is a potent inducer of the HMOX 1 gene transcriptionand has been shown to up-regulate it as much as eightfold overcontrol.15 Studies have also shown that when heme oxygenaseactivity is inhibited, the cytoprotective effect of CAPE against men-adione induced oxidative stress is abolished.15 Within a series ofcatechol ring-fluorinated CAPE analogs, cytoprotection is corre-lated with up-regulation of HMOX 1, but not direct anti-oxidantactivity.16 However, for CAPA and the CAPA analogs examinedhere, it is not clear how the balance of direct and indirect anti-oxi-dant effects contribute to cytoprotection. Work is currently beingdone to evaluate the role of HMOX 1 and to determine if CAPAexhibits similar response in regards to activation of HMOX 1.

The findings demonstrate that CAPA is less toxic than CAPE, andthat there is no significant difference in cytoprotection betweenthe two when tested at 5 and 20 lM concentrations against2 mM hydrogen peroxide. It is anticipated that CAPA is more stablethan CAPE in plasma. Previous studies have shown that ester com-pounds are very prone to hydrolysis via esterases, and that CAPEexhibits a short half life in plasma.16 Plasma stability is an impor-tant issue in the drug development process, as it determines howmuch of the initial dose actually reaches the target site. Cinnamicacid-derived amides are known to be particularly stable to hydro-lysis.33 In preliminary studies, the expected increased stability ofCAPA has been confirmed. CAPA is surprising stable to acid(0.1 M HCl, pH �1), although it undergoes hydrolysis readily atpH �10. While incubation of CAPE (100 lM) in rat plasma for18 h at 37 �C results in complete hydrolysis, CAPA remains largelyintact under these conditions (see Supplementary data).

4. Conclusions

CAPA and catechol ring-fluorinated derivatives of CAPA weresynthesized and investigated for cytoprotective activity againsthydrogen peroxide induced oxidative stress in HUVEC. All butone of the CAPA derivatives synthesized were non-toxic up tothe maximally tested concentration, with 4c being toxic at alltested concentrations. The results here also show that CAPA, 4b,4c, and 4e are all significantly cytoprotective in this model. Theonly two analogs which were not cytoprotective were the methyl-ated compounds 4d and 4f. Although the mechanism of cytopro-tection for these amides is not well understood, cytoprotection iscorrelated with the presence of free catechol hydroxyl groups inthe analogs examined. CAPA was less toxic in HUVEC when com-pared to CAPE, however, there was no significant difference foundin cytoprotection between the two compounds. This is significantas CAPA retains CAPE’s cytoprotective activity yet is more stablein plasma.

5. Experimental

5.1. Materials and apparatus

The reagents chloroacetyl chloride, phenethylamine, chloro-tert-butyldimethylsilane (TBDMSCl), 3,4-dihydroxybenzaldehyde,2-fluoro-4,5-dimethoxybenzaldehyde, 3-fluoro-4-methoxybenzal-

5036 J. Yang et al. / Bioorg. Med. Chem. 18 (2010) 5032–5038

dehyde, 3-fluoro-4-hydroxy-5-methoxybenzaldehyde, tetra-butyl-ammonium fluoride (TBAF), hydrogen peroxide, and boron tribro-mide were purchased from Sigma Aldrich (St Louis, MO) andused without further purification. All solvents were distilled priorto use. Nuclear magnetic resonance (NMR) spectroscopy was per-formed with a Varian Unity + 300 (300 MHz). Melting points wereobtained using a Buchi B-540 apparatus and are uncorrected. Massspectrometry services were provided by the Mass SpectrometryFacility at the University of Texas at Austin. Carbon, hydrogen,and nitrogen (CHN) elemental analysis was conducted by Quanti-tative Technologies Inc. (Whitehouse, NJ). HPLC was performedon a Varian Prostar 320 system. Purity of the final compoundswas assessed by both normal (Si) and reverse phase (C8) HPLC.

5.2. Preparation of Wittig reagent

5.2.1. 2-Chloro-N-phenethylacetamide (1)24,25

To a solution of phenethylamine (20 mmol, 2.52 mL) in CH2Cl2

(40 mL) was added K2CO3 (24 mmol, 3.32 g). Chloroacetylchloride(22 mmol, 1.75 mL) was slowly added to the reaction mixture.The reaction mixture was stirred at 45 �C under argon for 18 h.The mixture was diluted with CH2Cl2, washed with water andbrine, and then dried over Na2SO4. The resulting solution was con-centrated under a rotary evaporator and the resulting solid filteredto give 2.93 g of white crystals (74% yield); mp 63.6–64.6 �C (lit,25

65 �C); 1H NMR spectrum matches literature.25

5.2.2. Phenethylcarbamoylmethyl-triphenylphosphoniumchloride (2)

To a solution of triphenylphosphine (18.9 mmol, 4.96 g) in THF(50 mL) was added 2-chloro-N-phenethylacetamide (1) (12.6 mmol,2.5 g). The mixture was stirred at 85 �C for 72 h under argon. Thereaction mixture was diluted with diethyl ether and filtered, giving4.75 g of white solid (82% yield); mp 220.9–222.8 �C; 1H NMR(CDCl3) d: 2.67 (2H, t, J = 8.1 Hz), 3.31 (2H, q), 5.05 (2H, d,J = 14.4 Hz), 7.15–7.25 (5H, m), 7.59–7.68 (5H, m), 7.72–7.88 (10H,m). 13C NMR (CDCl3) d: 32.30 (JC–P = 54.94 Hz), 35.35, 41.73,118.63 (JC–P = 88.45 Hz), 126.33, 128.73 (JC–P = 30.79 Hz), 130.31(JC–P = 12.68 Hz), 134.24 (JC–P = 10.49 Hz), 135.16 (JC–P = 2.72 Hz),139.17, 162.661 (JC–P = 4.98 Hz); CI-MS m/z 424 (M�Cl�, 100).HRCI-MS: Calcd for C28H27NOP: 424.1830. Found: 424.1825.

5.3. General procedure for the demethylation of benzaldehydes

5.3.1. 2-Fluoro-4,5-dihydroxybenzaldehyde2-Fluoro-4,5-dimethoxybenzaldehyde (4 mmol, 736 mg) was

dissolved in 10 mL of CH2Cl2. The mixture was placed in a �78 �Cacetone and dry ice bath and 10 mL of a 1 M solution of BBr3 inCH2Cl2 was added slowly under argon. The reaction mixture wasallowed to warm to room temperature and stirred for 18 h.Methanol was added to the resulting mixture, and the solventevaporated. This process was repeated three times. Column chro-matography (5:1 CH2Cl2/EtOAc) afforded 590 mg (94.5% yield) of2-fluoro-4,5-dihydroxybenzaldehyde as a white solid which wascarried forward without further purification.

5.4. General procedure for the Wittig reaction

5.4.1. 3-(3,4-Dihydroxyphenyl)-N-phenethylacrylamide (4a,CAPA)

A mixture of 3,4-dihydroxybenzaldehyde (3 mmol, 414 mg),imidazole (9 mmol, 612 mg), TBDMSCl (9 mmol, 1356 mg), andDMAP (0.3 mmol, 36 mg) were dissolved in 5 mL of DMF andallowed to react at room temperature under argon for 1 h. Thereaction mixture was extracted with diethyl ether, washed withdeionized water, and then dried over Na2SO4. Column chromatog-

raphy (2% EtOAc in hexane) of the residue after evaporation of thesolvent afforded 540 mg of the protected benzaldehyde, which wascombined with the phosphonium chloride (2) (1.8 mmol, 828 mg)and Cs2CO3 (3.9 mmol, 1651 mg) and then 5 mL of dioxane and5 mL of CHCl3. The resulting mixture was heated to 60 �C for18 h. The reaction solution was separated, and the solid washedwith CHCl3. The combined organics were washed with water, driedover Na2SO4, and evaporated. Column chromatography (3:1 hex-ane/EtOAc) of the residue gave 550 mg of yellow oil. The oil wasdissolved in 5 mL of THF and TBAF (2.5 mL, 1 M in THF) was thenadded and the mixture was stirred for 5 min at 0 �C. The reactionmixture was concentrated on a rotary evaporator and subjectedto chromatography on a silica gel column (4:3 EtOAc/hexane).Recrystallization (CH2Cl2 and hexane) afforded 115 mg of 4a (14%overall yield from 3,4-dihydroxybenzaldehyde) as a white solid:mp 145 �C (lit,19 138–140 �C); 1H NMR matches literature; 13CNMR (CDCl3): d 35.48, 41.09, 113.89, 115.30, 117.17, 120.95,126.2, 127.13, 128.35, 128.65, 139.39, 141.07, 145.56, 147.60,168.14. CI-MS m/z 284 (MH+, 100). HRCI-MS: Calcd forC17H18NO3; 284.1287. Found: 284.1288.

The following compounds were prepared following the sameprocedure:

5.4.2. 3-(2-Fluoro-4,5-dihydroxyphenyl)-N-phenethylacrylamide(4b)

Recrystallization (CH2Cl2 and hexane) afforded 80 mg of 4b (7%overall yield from 2-fluoro-4,5-dihydroxybenzaldehyde) as a whitesolid: mp 145 �C; 1H NMR (DMSO-d6) d (ppm): 2.77 (t, J = 6.9 Hz,2H), 3.39 (q, J = 6.6 Hz, 2H), 6.40 (d, J = 15.9 Hz, 1H), 6.60 (d,J = 12.3 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 7.28 (m, 5H), 8.18 (s, 1H),9.17 (s, 1H), 9.89 (s, 1H); 13C NMR (DMSO-d6) d: 35.44, 41.11,102.81 (JC–F = 26 Hz), 113.07 (JC–F = 5 Hz), 113.29, 119.27(JC–F = 6 Hz), 126.20, 128.35, 128.64, 133.36, 139.37, 142.09,148.67 (JC–F = 11.55 Hz), 155.77 (JC–F = 243 Hz), 167.88; CI-MS m/z302 (MH+, 100). HRCI-MS: Calcd for C17H17NO3F; 302.1192. Found:302.1194.

5.4.3. 3-(3-Fluoro-4,5-dihydroxyphenyl)-N-phenethylacrylamide(4c)

Recrystallization (CH2Cl2 and hexane) afforded 135 mg of 4c(15% overall yield from 3-fluoro-4,5-dihydroxybenzaldehyde) as awhite solid: mp 154 �C; 1H NMR (DMSO-d6) d (ppm): 2.76 (d,J = 7.2 Hz, 2H), 3.39 (d, J = 6.6 Hz, 2H), 6.36 (d, J = 15.6 Hz, 1H),6.81 (d, J = 6 Hz, 1H), 6.86 (s, 1H), 7.23 (m, 5H), 8.09 (t, J = 5.1 Hz,1H), 9.46 (s, 1H), 9.71 (s, 1H); 13C NMR (DMSO-d6) d: 35.43, 41.1,106.41 (JC–F = 20 Hz), 110.59, 118.79, 126.10, 126.22, 128.36,128.64, 135.23, 139.34, 140.06, 147.57 (JC–F = 6 Hz), 152.28 (JC–F =238 Hz), 167.67; CI-MS m/z 302 (MH+, 100). HRCI-MS: Calcd forC17H17NO3F: 302.1192. Found: 302.1188.

5.4.4. 3-(3-Fluoro-4-hydroxy-5-methoxyphenyl)-N-phenethylacrylamide (4d)

An additional column chromatography purification (1:1.5EtOAc/hexane) to remove traces of the Z-isomer, afforded 100 mgof 4d (22% overall yield from 3-fluoro-4-hydroxy-5-methoxybenz-aldehyde) as a white foam: 1H NMR (CDCl3) d (ppm): 2.91 (t,J = 6.9 Hz, 2H), 3.68 (q, J = 6.2 Hz, 2H), 5.68 (s, 1H), 5.82 (s, 1H),6.20 (d, J = 15.4 Hz, 1H), 6.79 (s, 1H), 6.65 (dd, J = 1.6 Hz,J = 10.8 Hz, 1H), 7.28 (m, 5H), 7.50 (d, J = 15.4 Hz, 1H); 13C NMR(CDCl3) d: 31.92, 37.13, 52.74, 102.72, 104.76 (JC–F = 19 Hz),115.81, 122.65 (JC–F = 9 Hz), 122.87, 125.00, 125.09, 131.72(JC–F = 14 Hz), 135.13, 136.53, 144.69 (JC–F = 6 Hz), 147.02(JC–F = 242 Hz), 162.22; CI-MS m/z 316 (MH+, 100). HRCI-MS: Calcdfor C18H19NO3F; 316.1349. Found: 316.1351.

J. Yang et al. / Bioorg. Med. Chem. 18 (2010) 5032–5038 5037

5.4.5. 3-(3-Fluoro-4-hydroxyphenyl)-N-phenethyl-acrylamide(4e)

This process afforded 90 mg of 4e (8% overall yield from 3-flu-oro-4-hydroxybenzaldehyde) as a yellow foam with a 3:1 (E)-/(Z)-isomer ratio by 1H NMR: 1H NMR (CDCl3) d (ppm) major iso-mer: 2.91 (t, J = 6.9 Hz, 2H), 3.68 (q, J = 6.1 Hz, 2H), 5.66 (s, 1H),6.18 (d, J = 15.6 Hz, 1H), 7.00 (t, J = 8.5 Hz, 1H), 7.12 (d, J = 8.5 Hz,1H), 7.18 (d, J = 1.8 Hz, 1H), 7.28 (m, 5H), 7.52 (d, J = 15.6 Hz,1H); 13C NMR (CDCl3) d: 35.80, 41.30, 114.85 (JC–F = 19 Hz),118.31, 118.66, 125.50, 126.95 (JC–F = 20 Hz), 127.18, 128.92,128.99, 138.89, 140.85, 146.99 (JC–F = 14 Hz), 151.86 (JC–F =241 Hz), 167.13; CI-MS m/z 286 (MH+, 100). HRCI-MS: Calcd forC17H17NO2F; 286.1243. Found: 286.1242.

5.4.6. 3-(2-Fluoro-4,5-dimethoxyphenyl)-N-phenethyl-acrylamide(4f)

Recrystallization from EtOAc and hexane gave 48 mg of 4f (63%yield from 2-fluoro-4,5-dimethoxybenzaldehyde) as white crys-tals: mp 149 �C; 1H NMR (CDCl3) d (ppm): 2.92 (t, J = 6.9 Hz, 2H),3.68 (q, J = 6.5, 2H), 3.88 (d, J = 8.7, 6H), 5.90 (br s, 1H), 6.38 (d,J = 15.6 Hz, 1H), 6.64 (d, J = 12.0, 1H), 6.90 (d, J = 7.20 Hz, 1H),7.29 (m, 5H), 7.66 (d, J = 15.6 Hz, 1H); 13C NMR (CDCl3): d 35.93,41.09, 56.53 (JC–F = 9.96 Hz), 100.45 (JC–F = 28 Hz), 110.54 (JC–F =4 Hz), 114.01 (JC–F = 13 Hz), 121.15 (JC–F = 7 Hz), 126.77, 128.99(JC–F = 11 Hz), 134.03, 139.15, 145.67, 151.41 (JC–F = 10 Hz),156.54 (JC–F = 248 Hz), 166.40; CI-MS m/z 330 (MH+, 100). HRCI-MS: Calcd for C19H21NO3F; 330.1505. Found: 330.1506; elementalAnal. Calcd for C19H20NO3F: C, 69.29; H, 6.12; N, 4.25. Found: C,69.03; H, 6.12; N, 4.21.

5.5. Cell culture

Human umbilical vein endothelial cells (HUVEC) were obtainedfrom Lifeline Technologies (Walkersville, MD) and cultivated on75 cm2 1% gelatin coated culture flasks using MCDB 131 cell cul-ture media (Invitrogen, Carlsbad CA) supplemented with 2% fetalbovine serum, ascorbic acid, heparin, VEGF, hydrocortisone bFGFand heparin (Lifeline Technologies). The cells were grown to con-fluency at 37 �C in humidified atmosphere with 5% CO2. HUVECwere then treated with Trypsin/EDTA and subcultivated onto gela-tin coated 96 well multi-plates and used when confluent. Popula-tion doubling levels 2 through 5 were used in the describedexperiments.

5.6. Cytotoxicity assay

Stock CAPE and CAPE amide derivative solutions were dissolvedin DMSO then diluted in MCDB 131 tissue culture media for use inthe assays. Confluent HUVEC were treated with CAPE and theamide derivatives for 24 h at 37 �C at concentrations ranging from10 to 60 lM. Following the 24 h incubation, the media was re-placed with 10% CellTiter-Blue� Blue solution (Promega, MadisonWI). HUVEC were incubated for 2 h at 37 �C then analyzed for fluo-rescence. The readings were taken at 545 nm excitation and590 nm emission wavelengths on a Spectramax M2 microplatereader (Molecular Devices, Sunnyvale CA). Cell viability was calcu-lated from these fluorescence readings.

5.7. Cytoprotection assay

Confluent HUVEC were treated with CAPE and the amide deriva-tives for 5 h at 37 �C. After the 5 h incubation, the compounds wereremoved from the wells, and the cells were washed twice withMCDB 131 buffer. Stock hydrogen peroxide solution (50%, Sigma–Al-drich) was diluted in MCDB 131 buffer, and incubated in the cells fol-lowing the buffer wash. HUVEC were incubated in the hydrogen

peroxide for 1 h at 37 �C. The hydrogen peroxide was then removed.The cells were washed once with MCDB 131 media, and were thenincubated in complete MCDB 131 media for 18 h at 37 �C. Followingthe 18 h period, the cells were treated with 10% CellTiter-Blue� solu-tion and analyzed for viability. In the dose–response cytoprotectionassay, percent cytoprotection for each compound was calculated bysubtracting the average fluorescent reading of the negative control(HUVEC treated only with DMSO and hydrogen peroxide) from thefluorescent values of each well. This was then divided by the averagefluorescence of the positive control (HUVEC treated only withDMSO) to obtain percent cytoprotection.

5.8. Statistical analysis

Data are reported as means ± standard deviation as a percent-age of the control. Differences between the groups were first ana-lyzed by ANOVA, and then evaluated by the Tukey-Kramer posthoc analysis. O’Brien’s and Bartlett’s tests showed that varianceswere equal among groups. P <0.05 was considered significant. Allstatistical analysis was performed using the JMP program (SAS).

Acknowledgments

This project was supported by the US Army Institute of SurgicalResearch, the Robert Welch Foundation (F-1298 and H-F-0032) andthe TI-3D.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bmc.2010.05.080.

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